Effects of Gold Nanoparticle and Enzyme Precipitation on Enhancement of Surface Plasmon Resonance Immunoassay: A Super Sensitive Measurement of Anti- Glutamic Acid Decarboxylase Antibody

Introduction: In this study, colloidal gold nanoparticle and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance biosensor.

Methods: The colloidal gold nanoparticle was synthesized as described by Turkevitch et al., and their surface was firstly functionalized with HS(CH2)11(OCH2CH2)3COOH (OEG3¬-COOH) by self assembling technique. Thereafter, those OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti-IgG antibody (specific to the Fc portion of all human IgG subclasses) to form an enzyme-immunogold complex. Characterization was performed by several methods: UV-Vis absorption, dynamic light scattering (DLS), transmission electron microscopy (TEM) and FTIR. The as-prepared enzyme-immunogold complex has been applied in enhancement of SPR immunoassay. A sensor chip used in the experiment was constructed by using 1:10 molar ratio of HS(CH2)11(OCH2CH2)6COOH and HS(CH2)11(OCH2CH2)3OH. The capture protein, GAD65 (autoantigen) which is recognized by anti-GAD antibody (autoantibody) in the sera of insulin-dependent diabetes mellitus patients, was immobilized onto the 1:10 surface via biotin-streptavidin interaction.

Results and conclusions: In the research, we reported the influences of gold nanoparticle and enzyme precipitation on the enhancement of SPR signal. Gold nanoparticle showed its enhancement as being consistent with other previous studies, while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. As the results, anti-GAD antibody could be detected at pg/ml level which is far higher than that of commercial ELISA detection kit. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

Mammalian triokinase and dihydroxyacetone kinase are the same enzyme

It is widely accepted that the ATP-dependent phosphorylation of D-glyceraldehyde in the fructokinase pathway of fructose metabolism requires the enzyme “triokinase”. However, experimental data on this enzyme are remarkably scarce. The enzyme has been purified from a variety of sources and peptides derived from the pig kidney enzyme show high similarity to human dihydroxyacetone kinase – an enzyme which also has FMN cyclase activity in high manganese ion concentrations. The properties of the two enzymes are also highly similar. Therefore it is proposed that mammalian triokinase and dihydroxyacetone kinase are, in fact, the same enzyme. This has consequences for investigations of normal and aberrant fructose metabolism and for the teaching of biochemistry in medical and science courses.

Use of immunosorbent-purified antigens of Fasciola hepatica in enzyme immunoassays

A monoclonal antibody specific for the T1 tegumental antigen of Fasciola hepatica was used as a solid-phase immunosorbent for the purification of T1 antigen from homogenised mature F hepatica. Material fractionated by this technique was successfully used in enzyme-linked immunoassays to detect antibodies to F hepatica in sera from sheep and cattle. Species differences in response to infection by F hepatica were demonstrated.

An assay for angiotensin-converting enzyme using capillary zone electrophoresis

A sensitive and rapid method was developed for angiotensin-converting enzyme (ACE) activity determination by capillary zone electrophoresis. Hippuryl-View the MathML source-histidyl-View the MathML source-leucine, a synthetic tripeptide, was used as the ACE-specific substrate. Capillary zone electrophoresis was employed to separate the products of the enzymatic reaction and the ACE activity was determined by quantification of hippuric acid, a result of the enzymatic reaction on the tripeptide. The capillary electrophoresis was performed in a 27 cm × 75 μm i.d. fused-silica capillary using 200 mM boric acid–borate buffer (pH 9.0) as a run buffer with an applied voltage of 8.1 kV at a capillary temperature of 23°C. The electrophoresis was monitored at 228 nm. Each electrophoretic run requires only a nanoliter of the enzymatic reactant solution, at only 6 min, rendering a powerful tool for the ACE assay.

A facile method for synthesizing PVA/Au, PVA/Ag and PVA/AuAg nanocomposites

In this paper, we presented a facile way of preparing PVA/Au, PVA/Ag and PVA/AuAg nanocomposites through in situ synthesis of metal nanoparticles in PVA hydrogel network using a room temperature atmospheric pressure microplasma. This is the first time microplasma technology is used for the fabrication of hydrogel based nanocomposites. The materials synthesized have been characterized for their microstructure and antibacterial properties. The nanoparticles synthesized within the PVA hydrogel network are found to be better dispersed than those synthesized in water, and their size and shape are more uniform. The new approach has opened a new avenue towards multi-scale synthesis of green and multi-functional nanocomposites, which may find wide range of potential applications in biomedical field.

UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors

Leloir pathway enzyme uridine diphosphate (UDP)-galactose 4'-epimerase from the common liver fluke Fasciola hepatica (FhGALE) was identified and characterized. The enzyme can be expressed in, and purified from, Escherichia coli. The recombinant enzyme is active: the K(m) (470 μM) is higher than the corresponding human enzyme (HsGALE), whereas the k(cat) (2.3 s(-1)) is substantially lower. FhGALE binds NAD(+) and has shown to be dimeric by analytical gel filtration. Like the human and yeast GALEs, FhGALE is stabilized by the substrate UDP-galactose. Molecular modelling predicted that FhGALE adopts a similar overall fold to HsGALE and that tyrosine 155 is likely to be the catalytically critical residue in the active site. In silico screening of the National Cancer Institute Developmental Therapeutics Program library identified 40 potential inhibitors of FhGALE which were tested in vitro. Of these, 6 showed concentration-dependent inhibition of FhGALE, some with nanomolar IC50 values. Two inhibitors (5-fluoroorotate and N-[(benzyloxy)carbonyl]leucyltryptophan) demonstrated selectivity for FhGALE over HsGALE. These compounds also thermally destabilized FhGALE in a concentration-dependent manner. Interestingly, the selectivity of 5-fluoroorotate was not shown by orotic acid, which differs in structure by 1 fluorine atom. These results demonstrate that, despite the structural and biochemical similarities of FhGALE and HsGALE, it is possible to discover compounds which preferentially inhibit FhGALE.

Clinical potential of gene-directed enzyme prodrug therapy to improve radiation therapy in prostate cancer patients

Despite the advances in prostate cancer diagnosis and treatment, current therapies are not curative in a significant proportion of patients. Gene-directed enzyme prodrug therapy (GDEPT), when combined with radiation therapy, could improve the outcome of treatment for prostate cancer, the second leading cause of cancer death in the western world. GDEPT involves the introduction of a therapeutic transgene, which can be targeted to the tumour cells. A prodrug is administered systemically and is converted to its toxic form only in those cells containing the transgene, resulting in cell kill. This review will discuss the clinical trials which have investigated the potential of GDEPT at various stages of prostate cancer progression. The advantages of using GDEPT in combination with radiotherapy will be examined, as well as some of the recent advances which enhance the potential utility of GDEPT.

A nitrile-metabolising enzyme and a process for producing an acid using same

The present invention relates to an isolated nucleotide sequence and corresponding polypeptide derived from the nitrile-metabolising Pantoea strain deposited under NCIMB 41854. Said isolated polypeptide acts as a nitrilase and the invention extends to a process for producing a carboxylic acid using said isolated polypeptide to metabolise nitriles such as 3-hydroxyglutaronitrile, 3-hydroxybutyronitrile and 3- hydroxy-phenylpropionitrile to form corresponding carboxylic acids.